ABSTRACT Infections caused by MDR Pseudomonas aeruginosa and other Gram-negative pathogens challenge clinicians to find safe and effective antibiotic regimens that can eradicate these opportunistic pathogens from the frail, often immunocompromised hosts that they target. Two features of the P. aeruginosa cell envelope - its limited permeability to small molecules and the large number of both constitutive and inducible efflux systems it contains - render this pathogen intrinsically resistant to many available antimicrobials and contribute to acquired resistance toward the small set of existing anti-Pseudomonal antibiotics. This seriously limits the ability to identify ?hits? with antibiotic activity using whole-cell assays - as compounds that penetrate and can inhibit key metabolic pathways are often effluxed out before they measurably inhibit bacterial growth or viability. Approaches that identify novel small molecule inhibitors of key bacterial enzymes often fail when these small molecules cannot achieve effective intrabacterial concentrations - and our understanding of the chemistries that would allow for penetration and retention is woefully incomplete. In this application we use a diverse array of riboswitches, sensitive and specific RNA-based small molecule sensors, as rapid and quantitative indicators that bacterial physiology has been perturbed. By multiplexing several riboswitches that report on accumulation of the alarmones ZTP and ppGpp, as well as the toxic product of increased SAM utilization, SAH, we can effectively screen for ?signatures? of a bacterial response to sub- MIC levels of small molecules. Our approach places these riboswitch reporters in isogenic MDR and efflux- deficient P. aeruginosa strains, simultaneously yielding information about both physical and structural chemical features that allow penetration and efflux-avoidance and identifying ?hit? molecules that can be developed as leads for new antibacterial agents. Our medicinal chemistry approach will build on both types of knowledge, allowing novel anti-Pseudomonal compounds to be identified and optimized.